Human VEGF-specific oligonucleotides

ABSTRACT

Disclosed are methods of reducing neovascularization and of treating various disorders associated with neovascularization. These methods include administering to a tissue or subject a synthetic oligonucleotide specific for vascular endothelial growth factor nucleic acid effective in inhibiting the expression of vascular endothelial growth factor.

FUNDING

This invention was made with Government support under Grant No. RO1EY0869 awarded by the National Institutes of Health. Thus, theGovernment has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of patent application Ser. No.08/398,945, entitled "HUMAN VEGF-SPECIFIC OLIGONUCLEOTIDES", filed onMar. 2, 1995; which is a continuation-in-part of U.S. patent applicationSer. No. 08/378,860, entitled "INHIBITION OF NEOVASCULARIZATION USINGVEGF-SPECIFIC OLIGONUCLEOTIDES", filed Jan. 26, 1995; which is acontinuation-in-part of U.S. patent application Ser. No. 08/098,942,entitled "ANTISENSE OLIGONUCLEOTIDE INGIBITION OF VASCULAR ENDOTHELIALGROWTH FACTOR EXPRESSION", filed Jul. 27, 1993, and assigned to thepresent assignees.

BACKGROUND OF THE INVENTION

This invention relates to neovascularization. More specifically, thisinvention relates to treatment of disorders that are associated withneovascularization using oligonucleotides specific for vascularendothelial growth factor.

Neovascular diseases of the retina such as diabetic retinopathy,retinopathy of prematurity, and age-related macular degeneration are amajor cause of blindness in the United States and the world, yet thebiochemical events responsible for these processes have not been fullyelucidated.

Diabetic retinopathy is the leading cause of blindness among working ageadults (20-64) in the United States (Foster in Harrison's Principles ofInternal Medicine (Isselbacher et al., eds.) McGraw-Hill, Inc., New York(1994) pp. 1994-1995). During the course of diabetes mellitus, theretinal vessels undergo changes that result in not only leaky vesselsbut also vessel drop out resulting in retinal hypoxia. The effects ofthese complications are hemorrhaging, "cotton wool" spots, retinalinfarcts, and neovascularization of the retina resulting in bleeding andretinal detachment. If left untreated, there is a 60% chance of visualloss. Classic treatment for proliferative diabetic retinopathy ispanretinal laser photocoagulation (PRP). However, complications canoccur from panretinal laser photocoagulation such as foveal burns,hemorrhaging, retinal detachment, and choroidal vessel growth.Furthermore, other untoward effects of this treatment are decreasedperipheral vision, decreased night vision, and changes in colorperception (Am. J. Ophthalmol. (1976) 81:383-396; Ophthalmol. (1991)98:741-840).

Thus, there is a need for a more effective treatment for diabeticretinopathy.

Retinopathy of prematurity (ROP) is a common cause of blindness inchildren in the United States (Pierce et al. (1994) Int. Ophth. Clinics34:121-148). Premature babies are exposed to hyperoxic conditions afterbirth even without supplemental oxygen because the partial pressure ofoxygen in utero is much lower than what is achieved when breathingnormal room air. This relative hyperoxia is necessary for their survivalyet can result in ROP. The blood vessels of the retina cease to developinto the peripheral retina resulting in ischemia and localized hypoxicconditions as the metabolic demands of the developing retina increase.The resulting hypoxia stimulates the subsequent neovascularization ofthe retina. This neovascularization usually regresses but can lead toirreversible vision loss. There are at least 10,000 new cases per yearwith a worldwide estimate of 10 million total cases. At present, thereis no effective cure for ROP. Two therapeutic methods, cryotherapy andlaser therapy, have been used but are not completely effective andthemselves cause damage to the eye, resulting in a reduction of vision(Pierce et al. (1994) Int. Ophth. Clinics 34:121-148). Many otherantiangiogenic compounds have been tested, but no inhibition in retinalneovascularization has been reported (Smith et al. (1994) Invest.Ophthalmol. Vis. Sci. 35:1442; Foley et al. (1994) Invest. Ophthalmol.Vis. Sci. 35:1442).

Thus, there is a need for an effective treatment for ROP.

Age related macular degeneration is one of the leading causes ofblindness in older adults in the United States, and may account for upto 30% of all bilateral blindness among Caucasian Americans (Anonymous(1994) Prevent Blindness America). This disease is characterized by lossof central vision, usually in both eyes, due to damage to retinalpigment epithelial cells which provide physiological support to thelight sensitive photoreceptor cells of the retina. In most cases thereis currently no effective treatment. In approximately 20% of exudativecases that are diagnosed early, laser treatment can prevent further lossof vision; however, this effect is temporary (Bressler et al.,Principles and Practices of Ophthalmology (eds. Albert and Jakobiac), W.B. Saunders Co., Philadelphia, Pa.) (1994) Vol. 2 pp. 834-852).

Thus, there is a need for a more effective and permanent treatment forage related macular degeneration.

Ocular neovascularization is also the underlying pathology in sicklecell retinopathy, neovascular glaucoma, retinal vein occlusion, andother hypoxic diseases. These eye diseases as well as other pathologicalstates associated with neovascularization (i.e., tumor growth, woundhealing) appear to have hypoxia as a common factor (Knighton et al.(1983) Science 221:1283-1285; Folkman et al. (1987) Science 235:442-446;Klagsbrun et al. (1991) Ann. Rev. Physiol. 53:217-239; Miller et al.(1993) Principles and Practice of Ophtalmology, W. B. Saunders,Philadelphia, pp. 760; and Aiello et al. (1994) New Eng. J. Med.331:1480-1487). Moreover, retinal neovascularization has beenhypothesized to be the result of a "vasoformative factor" which isreleased by the retina in response to hypoxia (Michaelson (1948) Trans.Opthalmol. Soc. U.K. 68:137-180; and Ashton et al. (1954) Br. J.Ophthalmol. 38:397-432). Recent experimental data show a highcorrelation between vascular endothelial growth factor expression andretinal neovascularization (Aiello et al. (1994) New Eng. J. Med.331:1480-1487). Furthermore, elevated levels of vascular endothelialgrowth factor have recently been found in vitreous from patients withdiabetes (Aiello et al., ibid.). Thus, this cytokine/growth factor mayplay an important role in neovascularization-related disease.

Vascular endothelial growth factor/vascular permeability factor(VEGF/VPF) is an endothelial cell-specific mitogen which has recentlybeen shown to be stimulated by hypoxia and required for tumorangiogenesis (Sanger et al. (1986) Cancer 46:5629-5632; Kim et al.(1993) Nature 362:841-844; Schweiki et al. (1992) Nature 359:843-845;Plate et al. (1992) Nature 359:845-848). It is a 34-43 kDa (with thepredominant species at about 45 kDa) dimeric, disulfide-linkedglycoprotein synthesized and secreted by a variety of tumor and normalcells. In addition, cultured human retinal cells such as pigmentepithelial cells and pericytes have been demonstrated no secrete VEGFand to increase VEGF gene expression in response to hypoxia (Adamis etal. (1993) Biochem. Biophys. Res. Commun. 193:631-638; Plouet et al.(1992) Invest. Ophthalmol. Vis. Sci. 34:900; Adamis et al. (1993)Invest. Ophthalmol. Vis. Sci. 34:1440; Aiello et al. (1994) Invest.Ophthalmol. Vis. Sci. 35: 1868; Simorre-Pinatel et al. (1994) Invest.Ophthalmol. Vis. Sci. Sci. 35:3393-3400). In contrast, VEGF in normaltissues is relatively low. Thus, VEGF appears to play a principle rolein many pathological states and processes related to neovascularization.Regulation of VEGF expression in tissues affected by the variousconditions described above could therefore be key in treatment orpreventative therapies associated with hypoxia.

SUMMARY OF THE INVENTION

It is known that cells affected by hypoxia induce VEGF. It has now beendiscovered that synthetic oligonucleotides specific for the mRNA forVEGF can inhibit hypoxia-associated neovascularization. This informationhas been exploited to develop the present invention which includesmethods of reducing neovascularization and of treating disorders anddiseases related to neovascularization. As used herein, the term"neovascularization" refers to the growth of blood vessels andcapillaries.

In the methods of the invention, an amount of a syntheticoligonucleotide specific for vascular endothelial growth factor nucleicacid and effective in inhibiting the expression of vascular endothelialgrowth factor is administered to a neovascularized tissue. This tissuemay be a culture or may be part or the whole body of an animal such as ahuman or other mammal.

As used herein, the term "synthetic oligonucleotide" refers tochemically synthesized polymers of nucleotides covalently attached viaat least one 5' to 3' internucleotide linkage. In some embodiments,these oligonucleotides contain at least one deoxyribonucleotide,ribonucleotide, or both deoxyribonucleotides and ribonucleotides. Inanother embodiment, the synthetic oligonucleotides used in the methodsof the invention are from about 14 to about 28 nucleotides in length. Inpreferred embodiments, these oligonucleotides contain from about 15 toabout 25 nucleotides.

In some embodiments, the oligonucleotides may also be modified in anumber of ways without compromising their ability to hybridize tonucleotide sequences contained within the mRNA for VEGF. The term"modified oligonucleotide" as used herein describes an oligonucleotidein which at least two of its nucleotides are covalently linked via asynthetic linkage, i.e., a linkage other than a phosphodiester linkagebetween the 5' end of one nucleotide and the 3' end of anothernucleotide in which the 5' nucleotide phosphate has been replaced withany number of chemical groups.

In some embodiments, at least one internucleotide linkage of theoligonucleotide is an alkylphosphonate, phosphorothioate,phosphorodithioate, phosphate ester, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,and/or carboxymethyl ester.

The term "modified oligonucleotide" also encompasses oligonucleotideswith a modified base and/or sugar. In addition, unoxidized or partiallyoxidized oligonucleotides having a substitution in one nonbridgingoxygen per nucleotide in the molecule are considered to be modifiedoligonucleotides. Also considered as modified oligonucleotides areoligonucleotides having nuclease resistance-conferring bulkysubstituents at their 3' and/or 5' end(s) and/or various otherstructural modifications not found in vivo without human intervention.Other modifications include those which are internal or are at theend(s) of the oligonucleotide molecule and include additions to themolecule of the internucleoside phosphate linkages, and terminal ribose,deoxyribose and phosphate modifications which cleave, or crosslink tothe opposite chains or to associated enzymes or other proteins whichbind to the genome or RNA.

A method of treating retinopathy of prematurity (ROP) is provided. Thismethod comprises the step of administering to a subject afflicted withROP a therapeutic amount of an oligonucleotide specific for vascularendothelial growth factor nucleic acid and effective in inhibiting theexpression of vascular endothelial growth factor in the retina.

In another aspect of the invention, a method of treating diabeticretinopathy is provided. This method includes administering to a subjectafflicted with diabetic retinopathy a therapeutic amount of anoligonucleotide specific for vascular endothelial growth factor nucleicacid and effective in inhibiting the expression of VEGF in the retina.

In yet another aspect of the invention, a method of treating age-relatedmacular degeneration (ARMD) is provided, which includes comprising thestep of administering to a subject afflicted with ARMD a therapeuticamount of an oligonucleotide specific for vascular endothelial growthfactor nucleic acid effective in inhibiting the expression of VEGF inthe retina.

In some preferred embodiments of the methods of the invention describedabove, the oligonucleotide is administered locally (e.g., intraocularlyor interlesionally) and/or systemically. The term "local administration"refers to delivery to a defined area or region of the body, while theterm "systemic administration is meant to encompass delivery to thewhole organism by oral ingestion, or by intramuscular, intravenous,subcutaneous, or intraperitoneaI injection.

Another aspect of the invention includes pharmaceutical compositionscapable of inhibiting neovascularization and thus are useful in themethods of the invention. These compositions include a syntheticoligonucleotide which specifically inhibits the expression of vascularendothelial growth factor and a physiologically and/or pharmaceuticallyacceptable carrier.

The term "pharmaceutically acceptable" means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s). The term "physiologically acceptable" refersto a non-toxic material that is compatible with a biological system suchas a cell, cell culture, tissue, or organism.

Another aspect of the invention is assessment of the role of VEGF inneovascularization associated with disease states.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIG. 1 is a diagrammatic representation of the murine model for retinalneovascularization;

FIG. 2 is a graphic representation of the ability of oligonucleotides ofthe invention to inhibit neovascularization during retinopathy ofprematurity;

FIG. 3 is a diagrammatic representation of the ELISA used to test theability of human VEGF-specific oligonucleotides to inhibit theexpression of VEGF;

FIG. 4 is a graphic representation of the results of an ELISAdemonstrating the reduction in the expression of VEGF in human cells inthe presence of human VEGF-specific oligonucleotides of the invention;and

FIG. 5 is a graphic representation of the results of a Northern blotdemonstrating the reduction in the expression of VEGF by human cells inthe presence of varying concentrations of human VEGF-specificoligonucleotides of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. The issuedU.S. patents, allowed applications, and references cited herein arehereby incorporated by reference.

The present invention provides synthetic antisense oligonucleotidesspecific for VEGF nucleic acid which are useful in treating diseases anddisorders associated with neovascularization including retinalneovascularization.

Antisense oligonucleotide technology provides a novel approach to theinhibition of gene expression (see generally, Agrawal (1992) Trends inBiotech. 10:152; Wagner (1994) Nature 372:333-335; and Stein et al.(1993) Science 261:1004-1012). By binding to the complementary nucleicacid sequence (the sense strand), antisense oligonucleotide are able toinhibit splicing and translation of RNA. In this way, antisenseoligonucleotides are able to inhibit protein expression. Antisenseoligonucleotides have also been shown to bind to genomic DNA, forming atriplex, and inhibit transcription. Furthermore, a 17 mer base sequencestatistically occurs only once in the human genome, and thus extremelyprecise targeting of specific sequences is possible with such antisenseoligonucleotides.

It has been determined that the VEGF coding region is comprised of eightaxons (Tischer et al. (1994) J. Biol. Chem. 266:11947-11954). Three VEGFtranscripts, 121, 165, and 189 amino acids long, have been observed,suggesting that an alternative splicing mechanism is involved (Leung etal. (1989) Science 246:1306-1309; Tischer et al. (1991) J. Biol. Chem.266:11947-11954). More recently, a fourth VEGF transcript was discoveredwhich has a length encoding 206 amino acids (Houck et al. (1991) Mol.Endocrinol. 5:1806-1814). Transcripts analogous to the 121 and 165 aminoacid polypeptides have been identified in the bovine system (Leung etal. (1989) Science 246:1306-1309), and the transcript corresponding tothe 165 amino acid transcript have also been identified in the rodentsystem (Conn et al. (1990) Proc. Natl. Acd. Sci. (USA) 87:1323-1327);Senger et al. (1990) Cancer Res. 50:1774-1778; Claffey et al. (1992) J.Biol. Chem. 267:16317-16322). Nucleic acid sequences encoding threeforms of VEGF have also been reported in humans (Tischer et al. (1991)J. Biol. Chem. 266:11947-11954), and comparisons between the human andthe murine VEGF have revealed greater than 85% interspecies conservation(Claffey et al. (1992) J. Biol. Chem. 267:16317-16322).

The oligonucleotides of the invention are directed to any portion of theVEGF nucleic acid sequence that effectively acts as a target forinhibiting VEGF expression. The sequence of the gene encoding VEGF hasbeen reported in mice (Claffey et al., ibid.) and for humans (Tischer etal., ibid.). These targeted regions of the VEGF gene include anyportions of the known exons. In addition, exon-intron boundaries arepotentially useful targets for antisense inhibition of VEGF expression.

The nucleotide sequences of some representative, non-limitingoligonucleotides specific for human VEGF are listed below in TABLE 1.

                                      TABLE 1                                     __________________________________________________________________________         TARGETED                       SEQ ID                                    OLIGO                                                                              SITE   SEQUENCE (AS)           NO:                                       __________________________________________________________________________    H-1  21-2   5'-CGCCGGGCCGCCAGCACACT-3'                                                                             1                                        H-1R 21-2   5'-CGCCGGGCCGCCAGCACACU-3'                                                                             2                                        H-1A 16-2   5'-GGCCGCCAGCACACT-3'    3                                        H-1B 26-2   5'-GCTCGCGCCGGGCCGCCAGCACACT-3'                                                                        4                                        H-2  76-57  5'-CAAGACAGCAGAAAGTTCAT-3'                                                                             5                                        H-3  80-62  5'-CACCCAAGACAGCAGAAAG-3'                                                                              6                                        H-3A 80-66  5'-CACCCAAGACAGCAG-3'    7                                        H-3B 86-62  5'-CCAATGCACCCAAGACAGCAGAAAG-3'                                                                        8                                        H-4  64-45  5'-AAGTTCATGGTTTCGGAGGC-3'                                                                            10                                        H-5  62-43  5'-GTTCATGGTTTCGGAGGCCC-3'                                                                            11                                        H-6  138-119                                                                              5'-GTGCAGCCTGGGACCACTTG-3'                                                                            12                                        H-7  628-609                                                                              5'-CGCCTCGGCTTGTCACATCT-3'                                                                            13                                        H-8  648-629                                                                              5'-CTTCCTCCTGCCCGGCTCAC-3'                                                                            14                                        H-8R 648-629                                                                              5'-CUUCCUCCUGCCCGGCUCAC-3'                                                                            15                                        H-8A 648-634                                                                              5'-CTTCCTCCTGCCCGG-3'   16                                        H-8B 653-629                                                                              5'-GGCTCCTTCCTCCTGCCCGGCTCAC-3'                                                                       17                                        H-9  798-779                                                                              5'-GTCTCCTCTTCCTTCATTTC-3'                                                                            18                                        H-9A 798-784                                                                              5'-GTCTCCTCTTCCTTC-3'   19                                        H-9B 803-779                                                                              5'-GCAGAGTCTCCTCTTCCTTCATTTC-3'                                                                       20                                        H-10 822-803                                                                              5'-CGGACCCAAAGTGCTCTGCG-3'                                                                            21                                        H-10A                                                                              817-803                                                                              5'-CCAAAGTGCTCTGCG-3'   22                                        H-10B                                                                              827-803                                                                              5'-CCCTCCGGACCCAAAGTGCTCTGCG-3'                                                                       23                                        H-11 E1-I1  5'-GGGCACGACCGCTTACCTTG-3'                                                                            24                                        H-12 I1-E2  5'-GGGACCACTGAGGACAGAAA-3'                                                                            25                                        H-13 I2-E3  5'-CACCACTGCATGAGAGGCGA-3'                                                                            26                                        H-14 E3-I3  5'-TCCCAAAGATGCCCACCTGC-3'                                                                            27                                        H-15 I3-E4  5'-CGCATAATCTGGAAAGGAAG-3'                                                                            28                                        H-17 59-40  5'-CATGGTTTCGGAGGCCCGAC-3'                                                                            30                                        H-17B                                                                              59-40  5'-CAUGGTTUCGGAGGCCCGAC-3'                                                                            31                                        H-18 61-42  5'-TTCATGGTTTCGGAGGCCCG-3'                                                                            32                                        E1/I1                                                                              E1/I1  5'-GACCGCTTACCTTGGCATGG-3'                                                                            33                                        I1/E2                                                                              I1/E2  5'-CCTGGGACCACTGAGGACAG-3'                                                                            34                                        E2/I2                                                                              E2/I2  5'-GGGACTCACCTTCGTGATGA-3'                                                                            35                                        I2/E3                                                                              I2/E3  5'-GAACTTCACCACTGCATGAG-3'                                                                            36                                        E3/I3                                                                              E3/I3  5'-TCCCAAAGATGCCCACCTGC-3'                                                                            37                                        I3/E4                                                                              I3/E4  5'-GCATAATCTGGAAAGGAAGG-3'                                                                            38                                        E4/I4                                                                              E4/I4  5'-ACATCCTCACCTGCATTCAC-3'                                                                            39                                        E4/I4B                                                                             E4/I4  5'-ACATCCUCACCTGCAUUCAC-3'                                                                            40                                        I4/E5                                                                              I4/E5  5'-TTTCTTTGGTCTGCAATGGG-3'                                                                            41                                        E5/I5                                                                              E5/I5  5'-GGCCACTTACTTTTCTTGTC-3'                                                                            42                                        I5/E7                                                                              I5/E7  5'-CACAGGGACTGGAAAATAAA-3'                                                                            43                                        E7/I7                                                                              E7/I7  5'-GGGAACCAACCTGCAAGTAC-3'                                                                            44                                        I7/E8                                                                              I7/E8  5'-GTCACATCTGAGGGAAATGG-3'                                                                            45                                        VH   641-621                                                                              5'-CTGCCCGGCTCACCGCCTCGG-3                                                                            46                                        H-19 56-38  5'-GGTTTCGGAGGCCCGACCG-3'                                                                             50                                        __________________________________________________________________________

With the published nucleic acid sequences and this disclosure provided,those of skill in the art will be able to identify, with without undueexperimentation, other antisense nucleic acid sequences that inhibitVEGF expression. For example, other sequences targeted specifically tohuman VEGF nucleic acid can be selected based on their ability to becleaved by RNAse H.

The oligonucleotides of the invention are composed of ribonucleotides,deoxyribonucleotides, or a combination of both, with the 5' end of onenucleotide and the 3' end of another nucleotide being covalently linked.These oligonucleotides are at least 14 nucleotides in length, but arepreferably 15 to 28 nucleotides long, with 15 to 25 mers being the mostcommon.

These oligonucleotides can be prepared by the art recognized methodssuch as phosphoramidate or H-phosphonate chemistry which can be carriedout manually or by an automated synthesizer as described in Uhlmann etal. (Chem. Rev. (1990) 90:534-583).

The oligonucleotides of the invention may also be modified in a numberof ways without compromising their ability to hybridize to VEGF mRNA.For example, the oligonucleotides may contain other than phosphodiesterinternucleotide linkages between the 5' end of one nucleotide and the 3'end of another nucleotide in which the 5' nucleotide phosphodiesterlinkage has been replaced with any number of chemical groups. Examplesof such chemical groups include alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. Oligonucleotides with these linkages can beprepared according to known methods (see, e.g., Uhlmann et al. (1990)Chem. Rev. 90:543-583).

Other modifications include those which are internal or at the end(s) ofthe oligonucleotide molecule and include additions to the molecule ofthe internucleoside phosphate linkages, such as cholesteryl or diaminecompounds with varying numbers of carbon residues between the aminogroups and terminal ribose, deoxyribose and phosphate modificationswhich cleave, or crosslink to the opposite chains or to associatedenzymes or other proteins which bind to the genome. Examples of suchmodified oligonucleotides include oligonucleotides with a modified baseand/or sugar such as 2'-O-alkylated ribose, arabinose instead of ribose,or a 3',5'-substituted oligonucleotide having a sugar which, at both its3' and 5' positions is attached to a chemical group other than ahydroxyl group (at its 3' position) and other than a phosphate group (atits 5' position). Other modified oligonucleotides are capped with anuclease resistance-conferring bulky substituent at their 3' and/or 5'end(s), or have a substitution in one nonbridging oxygen per nucleotide.Such modifications can be at some or all of the internucleosidelinkages, as well as at either or both ends of the oligonucleotideand/or in the interior of the molecule.

The preparation of these modified oligonucleotides is well known in theart (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158).For example, nucleotides can be covalently linked using art-recognizedtechniques such as phosphoramidate, H-phosphonate chemistry, ormethylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Chem.Rev. 90:543-584; Agrawal et al. (1987) Tetrahedron. Lett. 28: (31):3539-3542); Caruthers et al. (1987) Meth. Enzymol. 154:287-313; U.S.Pat. No. 5,149,798). Oligomeric phosphorothioate analogs can be preparedusing methods well known in the field such as methoxyphosphoramidite(see, e.g., Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA)85:7079-7083) or H-phosphonate (see, e.g., Froehler (1986) TetrahedronLett. 27:5575-5578) chemistry. The synthetic methods described in Bergotet al. (J. Chromatog. (1992) 559:35-42) can also be used.

The synthetic antisense oligonucleotides of the invention in the form ofa therapeutic formulation are useful in treating diseases, anddisorders, and conditions associated with neovascularization including,but not limited to, retinal neovascularization, tumor growth, and woundhealing.

The synthetic oligonucleotides of the invention may be used as part of apharmaceutical composition when combined with a physiologically and/orpharmaceutically acceptable carrier. The characteristics of the carrierwill depend on the route of administration. Such a composition maycontain, in addition to the synthetic oligonucleotide and carrier,diluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials well known in the art. The pharmaceutical composition of theinvention may also contain other active factors and/or agents whichenhance inhibition of VEGF expression or which will reduceneovascularization. For example, combinations of syntheticoligonucleotides, each of which is directed to different regions of theVEGF mRNA, may be used in the pharmaceutical compositions of theinvention. The pharmaceutical composition of the invention may furthercontain nucleotide analogs such as azidothymidine, dideoxycytidine,dideosyinosine, and the like. Such additional factors and/or agents maybe included in the pharmaceutical composition to produce a synergisticeffect with the synthetic oligonucleotide of the invention, or tominimize side-effects caused by the synthetic oligonucleotide of theinvention. Conversely, the synthetic oligonucleotide of the inventionmay be included in formulations of a particular anti-VEGF oranti-neovascularization factor and/or agent to minimize side effects ofthe anti-VEGF factor and/or agent.

The pharmaceutical composition of the invention may be in the form of aliposome in which the synthetic oligonucleotides of the invention iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layerswhich are in aqueous solution. Suitable lipids for liposomal formulationinclude, without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like. Oneparticularly useful lipid carrier is lipofectin. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No.4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323. Thepharmaceutical composition of the invention may further includecompounds such as cyclodextrins and the like which enhance delivery ofoligonucleotides into cells, as described by Zhao et al. (in press), orslow release polymers.

As used herein, the term "therapeutically effective amount" means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,healing of chronic conditions characterized by neovascularization or areduction in neovascularization, itself, or in an increase in rate ofhealing of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of one or more of the syntheticoligonucleotide of the invention is administered to a subject afflictedwith a disease or disorder related to neovascularization, or to a tissuewhich has been neovascularized. The synthetic oligonucleotide of theinvention may be administered in accordance with the method of theinvention either alone of in combination with other known therapies forneovascularization. When co-administered with one or more othertherapies, the synthetic oligonucleotide of the invention may beadministered either simultaneously with the other treatment(s), orsequentially. If administered sequentially, the attending physician willdecide on the appropriate sequence of administering the syntheticoligonucleotide of the invention in combination with the other therapy.

Administration of the synthetic oligonucleotide of the invention used inthe pharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asintraocular, oral ingestion, inhalation, or cutaneous, subcutaneous,intramuscular, or intravenous injection.

When a therapeutically effective amount of synthetic oligonucleotide ofthe invention is administered orally, the synthetic oligonucleotide willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%synthetic oligonucleotide and preferably from about 25 to 90% syntheticoligonucleotide. When administered in liquid form, a liquid carrier suchas water, petroleum, oils of animal or plant origin such as peanut oil,mineral oil, soybean oil, sesame oil, or synthetic oils may be added.The liquid form of the pharmaceutical composition may further containphysiological saline solution, dextrose or other saccharide solution, orglycols such as ethylene glycol, propylene glycol or polyethyleneglycol. When administered in liquid form, the pharmaceutical compositioncontains from about 0.5 to 90% by weight of the syntheticoligonucleotide and preferably from about t to 50% syntheticoligonucleotide.

When a therapeutically effective amount of synthetic oligonucleotide ofthe invention is administered by intravenous, subcutaneous,intramuscular, intraocular, or intraperitoneal injection, the syntheticoligonucleotide will be in the form of a pyrogen-free, parenterallyacceptable aqueous solution. The preparation of such parenterallyacceptable solutions, having due regard to pH, isotonicity, stability,and the like, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, subcutaneous, intramuscular,intraperitoneal, or intraocular injection should contain, in addition tothe synthetic oligonucleotide, an isotonic vehicle such as SodiumChloride Injection, Ringer's Injection, Dextrose Injection, Dextrose andSodium Chloride Injection, Lactated Ringer's Injection, or other vehicleas known in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The amount of synthetic oligonucleotide in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patent has undergone. Ultimately, the attendingphysician will decide the amount of synthetic oligonucleotide with whichto treat each individual patient. Initially, the attending physicianwill administer low doses of the synthetic oligonucleotide and observethe patient's response. Larger doses of synthetic oligonucleotide may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 10 μgto about 20 mg of synthetic oligonucleotide per kg body or organ weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. Ultimately the attending physicianwill decide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.

Some diseases lend themselves to acute treatment while others require tolonger term therapy. Proliferative retinopathy can reach a threshold ina matter of days as seen in ROP, some cases of diabetic retinopathy, andneovascular glaucoma. Premature infants are at risk forneovascularization around what would be 35 weeks gestation, a few weeksafter birth, and will remain at risk for a short period of time untilthe retina becomes vascularized. Diabetic retinopathy can be acute butmay also smolder in the proliferative phase for considerably longer.Diabetic retinopathy will eventually become quiescent as thevasoproliferative signal diminishes with neovascularization ordestruction of the retina.

Both acute and long term intervention in retinal disease are worthygoals. Intravitreal injections of oligonucleotides against VEGF can bean effective means of inhibiting retinal neovascularization in an acutesituation. However for long term therapy over a period of years,systemic delivery (intraperitoneal, intramuscular, subcutaneous,intravenous) either with carriers such as saline, slow release polymers,or liposomes should be considered.

In some cases of chronic neovascular disease, systemic administration ofoligonucleotides may be preferable. Since the disease process concernsvessels which are abnormal and leaky, the problem of passage through theblood brain barrier may not be a problem. Therefore, systemic deliverymay prove efficacious. The frequency of injections is from continuousinfusion to once a month, depending on the disease process and thebiological half life of the oligonucleotides.

In addition to inhibiting neovascularization in vivo, antisenseoligonucleotides specific for VEGF are useful in determining the role ofthis cytokine in processes where neovascularization is involved. Forexample, this technology is useful in in vitro systems which mimic bloodvessel formation and permeability, and in in vivo system models ofneovascularization, such as the murine model described below.

A murine model of oxygen-induced retinal neovascularization has beenestablished which occurs in 100% of treated animals and is quantifiable(Smith et al. (1994) Invest. Ophthalmol. Vis. Sci. 35:101-111). Usingthis model, a correlation has been determined between increasingexpression of VEGF message and the onset of retinal neovascularizationin the inner nuclear and ganglion cell layers (i.e., in Muller cells)(Pierce et al. (1995) Proc. Natl. Acad. Sci. (USA) (in press). Thisresult has been confirmed by Northern blot and in situ hybridizationanalysis of whole retinas at different time points during thedevelopment of neovascularization (Pierce et al., ibid.).

That VEGF plays a role in retinal neovascularization has been shownusing the murine model of neovascularization described above. Threeindependent experiments were performed using antisense oligonucleotidesspecific for VEGF (JG-3 (SEQ ID NO 47), JG-4, (SEQ ID NO:48), and Vm(SEQ ID NO:46), and a corresponding sense oligonucleotide (V2 (SEQ IDNO:49). These oligonucleotides were designed using the known nucleicsequence of murine VEGF (Claffee et al. (1992) Biol. Chem.267:16317-16322). The sequence of the Vm oligonucleotide (SEQ ID NO:6)is targeted to the sequence surrounding the translational TGA stop site(TGA). The sequence of JG-4 (SEQ ID NO:48) is targeted to the sequence5' to and containing the ATG of the translational start site of themurine VEGF molecule. The sequence of JG-3 (SEQ ID NO:47) is targeted tothe 5' untranslated region, and the V2 sense sequence is targeted to thesequence surrounding the translational start site (ATG).

A compilation of the results of these experiments is presented in FIG.2. These results indicate that Vm (SEQ ID NO:46) antisenseoligonucleotide significantly reduces retinal neovascularization whencompared with both untreated and sense oligonucleotide V2, (SEQ IDNO:49) controls. JG-3 (SEQ ID NO:47) and JG-4 (SEQ ID NO:47) showsignificant activity when compared against untreated eyes. The sensecontrol oligonucleotide V2 (SEQ ID NO:49) does not show any significantactivity when compared with untreated eyes.

In the studies described above, the human VEGF antisense oligonucleotidewhich corresponds to murine JG-3 is H-1 (SEQ ID NO:1), which is targetedto the 5' untranslated region; that which corresponds to murine JG-4 isH-17 (SEQ ID NO:30), which is targeted to the sequence 5' to andcontaining the ATG of the translational start site of the human VEGFmolecule; and that which corresponds to the murine Vm gene is VH (SEQ IDNO:46), which is targeted to sequences surrounding the translationalstop site (TGA) of the human VEGF molecule. These antisenseoligonucleotides of the invention are expected to inhibit VEGFexpression in human cells in much the same way as the murine antisenseoligonucleotides inhibit expression of VEGF in mouse cells.

Human VEGF antisense sequences corresponding to other murine sequencesare also known. For example, human oligonucleotide H-6 (SEQ ID NO:12)corresponds to a region spanning murine sequences JG-6 (SEQ ID NO:52)and JG-7 (SEQ ID NO:53), and human oligonucleotide H-2 (SEQ ID NO:5) isin the same region as murine sequence JG-5 (SEQ ID NO:51). It is likelythat these sequences have a similar effect on inhibition of VEGFexpression and hence on controlling neovascularization.

There are several methods by which the effects of antisenseoligonucleotides on VEGF expression and neovascularization can bemonitored. One way is a capture ELISA developed for quantifying humanVEGF protein expressed by cells. Using this assay, it has beendetermined that an antisense phosphorothioate oligonucleotide H-3 (SEQID NO:6) targeted to a sequence just 3' to the translational start sitecan inhibit the hypoxic induction of VEGF expression in asequence-specific manner, compared with random (R) and sense (H-16, SEQID NO:29) controls), as shown in FIG. 4. This inhibition is reproducibleand in this in vitro system appears to be lipid carrier-specific andantisense-specific as only antisense oligonucleotide H-3 (SEQ ID NO:6)in the presence of lipofectin (a lipid carrier), and not lipofectamine(another lipid carrier), results in inhibition of VEGF proteinexpression.

At the RNA level, Northern blots (Sambrook et al. (1989) MolecularCloning; a Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork, Vol. 1, pp. 7.38; Arcellana-Panlilio et al. (1993) Meth. Enz.225:303-328) can be performed to determine the extent thatoligonucleotides of the invention inhibit the expression of VEGF mRNA.For example, as shown in FIG. 5, a histogram representing Northern blotanalysis demonstrates a decrease in VEGF RNA levels in culture humancells treated with antisense oligonucleotide H-3 (SEQ ID NO:6), whilethere is only a minimal change in VEGF RNA levels in samples treatedwith sense control H-16 (SEQ ID NO:29).

In addition, bioactivity can be determined by several methods, includingthe Miles vessel permeability assay (Miles and Miles (1952) J. Physiol.Lond.) 118:228), which measures vessel permeability, endothelial cellmitogenicity, which measures cell growth, and intracellular calciumrelease in endothelial cells (see, e.g., Brock and Capasso (1988) J.Cell. Physiol. 136:54), which measures the release of calcium inresponse to VEGF binding to its receptor on endothelial cells.

The following examples illustrate the preferred modes of making andpracticing the present invention, but are not meant to limit the scopeof the invention since alternative methods may be utilized to obtainsimilar results.

EXAMPLE 1 PREPARATION OF VEGF-SPECIFIC OLIGONUCLEOTIDES

Human VEGF cDNA is transcribed in vitro using an in vitro eukaryotictranscription kit (Stratagene, La Jolla, Calif.). The RNA is labelledwith ³² P using T-4 polynucleotide kinase as described by (Sambrook etal. (1989) Molecular Cloning; a Laboratory Manual, Cold Spring HarborLaboratory Press, New York, Vol. 1, pp. 5.71). The labelled RNA isincubated in the presence of a randomer 20 mer library and RNAse H, anenzyme which cleaves RNA-DNA duplexes (Boehringer Mannheim,Indianapolis, Ind.). Cleavage patterns are analyzed on a 6%polyacrylamide urea gel. The specific location of the cleaved fragmentsis determined using a human VEGF sequence ladder (Sequenase Kit, UnitedStates Biochemical, Cleveland, Ohio).

EXAMPLE 2 ANIMAL MODEL OF RETINAL NEOVASCULARIZATION

A. Preparation of Oligonucleotides

Synthesis of the following oligonucleotides: JG-3 (SEQ ID NO:47), JG-4(SEQ ID NO:48), Vm (SEQ ID NO:46), and V2 (SEQ ID NO:49), was performedon a Pharmacia Gene Assembler series synthesizer using thephosphoramidite procedure (see, e.g., Uhlmann et al. (Chem. Rev. (1990)90:534-583). Following assembly and deprotection, oligonucleotides wereethanol precipitated twice, dried, and suspended in phosphate-bufferedsaline (PBS) at the desired concentration.

The purity of these oligonucleotides was tested by capillary gelelectrophoreses and ion exchange HPLC. Endotoxin levels in theoligonucleotide preparation was determined using the Luminous AmebocyteAssay (Bang (1953) Biol. Bull. (Woods Hole, Mass.) 105:361-362).

B. Preparation of Animal Model

Seven day postnatal mice (P7, C57b1/6J, (Children's Hospital BreedingFacilities, Boston, Mass.) were exposed to 5 days of hyperoxicconditions (75±2%) oxygen in a sealed incubator connected to a Bird 3-Moxygen blender (flow rate: 1.5 liters/minute; Bird, Palm Springs,Calif.). The oxygen concentration was monitored by means of an oxygenanalyzer (Beckman, Model D2, Irvine, Calif.). After 5 days (P12), themice were returned to room air. Maximal retinal neovascularization wasobserved 5 days after return to room air (P17). After P21, the level ofretinal neovascularization was just beginning to regress.

C. Treatment

After mice had been removed from oxygen, antisense oligonucleotides wereinjected into the vitreous with a Hamilton syringe and a 33 gauge needle(Hamilton Company, Reno, Nev.). The animals were anesthetized for theprocedure with Avertin ip. The mice were given a single injection ofantisense oligonucleotides (or sense or non-sense controls) at P12achieving a final concentration of approximately 30 μM. The animals weresacrificed at P17 with tribromoethanol ip (0.1 ml/g body weight) andcervical dislocation.

D. Microscopy

The eyes were enucleated, fixed in 4% paraformaldehyde, and embedded inparaffin. Serial sections of the whole eyes were cut sagittally, throughthe cornea, and parallel to the optic nerve. The sections were stainedwith hematoxylin and periodic acid-Schiff (PAS) stain. The extent ofneovascularization in the treated eyes was determined by countingendothelial cell nuclei extending past the internal limiting membraneinto the vitreous. Nuclei from new vessels and vessel profiles could bedistinguished from other structures in the retina and counted incross-section with light microscopy. Additional eyes were sectioned andexamined by in situ hybridization to a VEGF probe.

To examine the retinal vasculature using fluorescein-dextran, the micewere perfused with a 50 mg/ml solution of high molecular weightfluorescein-dextran (Sigma Chemical Company, St. Louis, Mo.) in 4%paraformaldehyde. The eyes were enucteated, fixed in paraformaldehyde,and flat-mounted with glycerol-gelatin. The flat-mounted retinas wereviewed and photographed by fluorescence microscopy using an Olympus BX60fluorescence microscope (Olympus America Corp., Bellingham, Mass.).

EXAMPLE 3 RETINOPATHY OF PREMATURITY

A. Preparation of Oligonucleotides

VEGF specific oligonucleotides are synthesized as described in EXAMPLE2A above. Sterile and endotoxin-free oligonucleotides are diluted inBalanced Salt Solution (BSS, Alcon, Fort Worth, Tex.) so as to have thesame pH and electrolyte concentration as the aqueous or vitreous of theeye. Emalphor EC620 (2.5%, GAF Corp.) (Bursell et al. (1993) J. Clin.Invest. 92:2872-2876), a petroleum product, is added to change viscosityand aid in delivery properties. Doses to achieve intravitrealconcentrations ranging from 0.1 μM-100 μM are administered depending onthe severity of the retinal/ocular neovascularization. The volumedelivered is between 1 μl and 1 ml depending on the volume of the eye.

B. ROP Patient Profile

The patient treated is a premature, 34 week post-conception Caucasianfemale weighing less than 1,000 grams at birth and isrespirator-dependent. The patient has bilateral stage 3+ disease with 11clock hours of neovascularization in each eye. There is hemorrhaging inone eye, and both eyes have reached "threshold" according to theinternational classification (i.e., each eye has >50% chance of going onto retinal detachment). Extraretinal fibrovascular proliferation isfound in both eyes.

C. Treatment

The intubated patient is Anesthetized with fluorane. The face and eyesare prepared with a betadine scrub and draped in the usual sterilefashion. The sterile drug with vehicle is injected with a 33 gaugeneedle on a sterile syringe at the posterior limbus (pars plana) throughfull thickness sclera into the vitreous. No closing suture is requiredunless there is leakage. Antibiotic drops containing gentamicin orerythromycin ointment is applied to the surface of the globe in thepalpebral fissure several times per day until there is complete woundclosure. The frequency of injection ranges from every other day to onceevery 6 months or less, depending on the severity of the diseaseprocess, the degree of intraocular inflammation, the character of thevehicle (i.e., slow release characteristics), the degree of inhibitionof the neovascularization and the tolerance of the eye to injections.Short and long term follow-up check-ups for possible retinal detachmentfrom the neovascular disease as well as from the injections arenecessary.

D. Monitoring of Progress

The eye upon dilation is monitored for signs of inflammation, infection,and resolution of neovascularization by both a direct and a indirectophthalmoscope to view the retina and fundus. A slit lamp exam is usedin some cases of anterior segment disease. Positive response totreatment includes fewer neovascular tufts, fewer clock hours ofinvolvement, and less tortuosity of large blood vessels. Monitoring canbe as frequent as every day in cases where premature infants arethreatened with retinal detachment from proliferative ROP. The frequencyof monitoring will diminish with resolution of neovascularization.

EXAMPLE 4 DIABETIC RETINOPATHY

A. Preparation of Oligonucleotides

VEGF specific oligonucleotides are synthesized as described in EXAMPLE2A above and prepared for administration as described in EXAMPLE 3Aabove. Doses to achieve intravitreal concentrations ranging from 0.1-100μM are administered depending on the severity of the retinal/ocularneovascularization. The volume delivered is between 1 μl and 1 mldepending on the volume of the eye and whether vitreous has beenpreviously removed as during a vitrectomy for diabetic eye disease.

B. Diabetic Patient Profile

The patient to be treated is a 30 year old African American malesuffering for 25 years from juvenile-onset diabetes. The patient hasbilateral proliferative retinopathy with sub-retinal hemorrhaging,cotton wool spots, and exudates. Upon fluorescein angiography, there arewell defined areas of neovascularization bilaterally with areas ofcapillary drop-out.

C. Treatment

The patient is treated weekly with intraocular injections ofoligonucleotides resuspended in the appropriate vehicle (BSS, Emanfour)at concentrations within the range of 0.1-100 μM. The treatment may besupplemented with systemic delivery of oligonucleotide (i.e.,intravenous, subcutaneous, or intramuscular) from 2 to 5 times per dayto once a month, depending on the disease process and the biologicalhalf life of the oligonucleotides.

D. Monitoring of Progress

The patient's eyes are monitored as described above in EXAMPLE 2D. Theeyes upon dilation are examined for regression of neovascularizationwith both a direct and an indirect ophthalmoscope to view the retina andfundus. A slit lamp exam is used in the case of anterior segmentdisease. Repeat injections are given as needed, based on the degree ofinhibition of the neovascularization and the tolerance of the eye toinjections. Short and long term follow-up check-ups are given to checkfor possible retinal detachment from the neovascular disease as well asfrom the injections.

EXAMPLE 5 AGE-RELATED MACULAR DEGENERATION

A. Preparation of Oligonucleotides

VEGF specific oligonucleotides are synthesized as described in EXAMPLE2A above and prepared as described in EXAMPLE 3A above. Doses to achieveintravitreal concentrations ranging from 1 μl and 1 ml are administereddepending on the severity of the retinal/ocular neovascularization. Thevolume delivered is between 1 μl and 1 ml depending on the volume of theeye and whether vitreous has been previously removed.

B. ARMD Patient Profile

The patient is a 50 year old Caucasian male suffering from the exudativeform of age related macular degeneration. This patient has choroidalneovascularization which is apparent from fluorescein angiography. Thedisease is bilateral and the patient has a reduction in vision in eacheye from 20/60 to 20/100.

C. Treatment

The patient is treated weekly with intraocular injections ofoligonucleotide resuspended in the appropriate vehicle (BSS, Emanfour)at concentrations within the range of 0.1 to 100 μM. This treatment maybe supplemented with systemic delivery of oligonucleotide (i.e.,intravenous, subcutaneous, or intramuscular) from 2 to 5 times per dayto once a month.

D. Monitoring of Progress

The eyes upon dilation are examined for regression of neovascularizationwith both a direct and an indirect ophthalmoscope to view the retina andfundus. Fluorescein angiography is used to check for the resolution ofneovascularization. A slit lamp exam is used in the case of anteriorsegment disease. Repeat injections are given as needed, based on thedegree of inhibition of the neovascularization and the tolerance of theeye to injections. Short and long term follow-up check-ups are given tocheck for possible retinal detachment from the neovascular disease aswell as from the injections.

EXAMPLE 6 HUMAN CELL CULTURE

U373 human neuroblastoma cells were cultured in Dulbecco's modifiedEarls (DME) medium containing glucose (4500 mg/ml) and glutamate (2 mM)(Mediatech, Washington, D.C.) supplemented with penicillin/streptomycin(100 IU/MI/100 mcg/ml, Mediatech, Washington, D.C.). The cells werecultured at 37° C. under 10% CO₂. The cells were plated in 96 welltissue culture dishes (Costar Corp., Cambridge, Mass.) and maintained asabove. The cells were placed under anoxic conditions for 18-20 hoursusing an anaerobic chamber (BBL Gas Pak, Cockeysville, Md.).

EXAMPLE 7 NORTHERN BLOTTING

In order to determine the level at which inhibition of VEGF expressionoccurs in cells in the presence of an oligonucleotide of the invention,Northern blotting was carried out. Human U373 cells cultured asdescribed in EXAMPLE 6 above were plated in 100 mm tissue culture dishesand treated for 12 hours in the presence of 5 μg/ml lipofectin(Gibco-BRL, Gaithersburg, Md.) as a lipid carrier with oligonucleotideH-3 (SEQ ID NO:6) (antisense oligonucleotide) and H-16 (SEQ ID NO:29)(sense oligonucleotide) at 0.05 μM, 0.5 μM, and 2.0 μM, respectively.The cells were refed after 12 to 15 hours with freshmedia+oligonucleotide (minus lipofectin) and allowed to recover for 5 to7 hours. The cells were placed in hypoxia for 18 to 20 hours total RNAwas isolated using the single-step acid guanidiniumthiocyanate-phenol-chloroform extraction method described by Chomczynskiand Sacchi (Anal. Biochem. (1987) 162:156-159). Northern blotting wasperformed according to the methods of Sambrook et al. (MolecularCloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork) (1989) Vol. 1, pp. 7.38) or Arcellana-Panlilio et al. (Meth. Enz.(1993) 225:303-328). All RNA signals were quantified on a Phosphorimager(BioRad, Hercules, Calif.) and normalized using the 36B4 cDNA probe(Laborda (1991) Nucleic Acids Res. 19:3998).

EXAMPLE 8 ELISA VEGF PROTEIN STUDY

U373 neuroblastoma cells as described in EXAMPLE 6 above were plated ina 96 well tissue culture dish and treated overnight with varyingconcentrations of antisense oligonucleotides against human VEGF in thepresence of 5 μg/ml lipofectin. The cells were refed after 12 to 15hours with fresh media+oligonucleotide (no lipofectin) and allowed torecover for 5 to 7 hours. The dishes were placed under hypoxicconditions for 18 to 20 hours using an anaerobic chamber (Gas Pac,Cockeysville, Md.). The media was analyzed using the antigen captureELISA assay described above (approximately 36 hours post treatment). Thehuman VEGF oligonucleotides used were H-3 (SEQ ID NO:6) (antisense,coding), H-16 (SEQ ID NO:29) (start site/coding, sense control), and arandom control (R).

The culture medium from the cells described in EXAMPLE 5 was analyzedfor VEGF protein as follows. 96-well plates (Maxizorb ELISA Nunc A/S,Camstrup, Denmark) were treated overnight at 4° C. with 100 μl/well ofthe capture antibody, a monoclonal antibody against human VEGF (R&DSystems, Minneapolis, Minn., 2.5 μg/ml in 1× PBS). The wells were washedthree times with 1×PBS/0.05% Tween-20 (United States Biochemical,Cleveland, Ohio) using a plate washer (Dynatech, Gurnsey ChannelIslands). Non-specific binding 5 sites in the wells were blocked byadding 2% normal human serum (100 μl) and incubating the plate at 37° C.for 2 hours. This blocking solution was removed and 200 μl conditionedmedium containing human VEGF added to each well and incubated at 37° C.for 2 to 3 hours. The plates were washed as described above. 100 μl ofthe primary antibody (618/619, 2 μg/ml in normal human serum) was addedto each well and incubated at 37° C. for 1 to 2 hours. The secondaryantibody was an affinity purified rabbit anti- human VEGF polyclonal).The plates were washed as described above. 100 μl of the detectionantibody, a horse radish peroxidase-labelled goat anti-mouse IgGmonoclonal antibody (1:10,000, Vector Laboratories, Burlinggame,Calif.), was added to each well and incubated at 37° C. for 1 hour. Theplates were washed as described above. The wells were developed usingthe TMB microwell peroxidase developing system (Kirkegaard and Perry,Gaithersburg, Md.), and quantified at 450 nm using a Ceres 900 platereader (Bio-Tek Instruments, Inc., Winooski, Vt.). The linear range ofthis assay is between 2 ng and 0.01 ng human VEGF. Representativeresults are shown in FIG. 3.

EXAMPLE 9 BIOACTIVITY ASSAYS

Bioactivity can be determined by the Miles vessel permeability assay(Miles and Miles (1952) J. Physiol. (Lond.) 118:228). Briefly, Hartleyguinea pigs (800 g) are shaved and depilated and injected intravenouslywith 1.0 ml of normal saline containing 0.5 g of Evans Blue dye per 100ml. Subcutaneous injections (250 μl) of serum-free medium containingunknown quantities of VEGF are performed. Positive (purified VEGF) andnegative controls (normal saline) are also included in the experiment.Twenty minutes post-injection, the animals are sacrificed and the testand control sites are cut out and quantitated for extravasation of EvansBlue dye. The limit of detection for this assay is 500 pM. Endothelialcell mitogenicity can also manifest bioactivity. In this method, humanumbilical vein endothelial (HUVEC) are grown and maintained using theBiocoat endothelial cell growth environment (Collaborative BiomedicalProducts, Bedford, Mass.). 1×10⁴ cells are then plated in duplicate on35 mM tissue culture dishes in 1.4 ml E-STIM medium (CollaborativeBiomedical Products, Bedford, Mass.) plus 5% heat-inactivated fetalbovine serum. Following cell attachment (about 4 hours), two dishes ofcells are trypsinized, counted, and used for a starting cell number.Test samples containing unknown amounts of VEGF are then added induplicate to the remaining dishes at day 0 and at day 2. Controlsconsisting of purified VEGF (positive) and PBS (negative) are also used.On day 4, the dishes of cells are trypsinized, counted and compared tothe 5 starting cell number. The limit of detection for this assay is 10pM.

The intracellular calcium release assay is also used to monitorbioactivity (see, e.g., Brock and Capasso (1988) J. Cell. Physiol.136:54). Human umbilical vein endothelial cells (HUVEC) are maintainedin EGM-UV medium. Cells are removed from the plate by means of EDTA andcollagenase. The calcium-sensitive dye, Fura-2 (Molecular Probes,Eugene, Oreg.), is used to monitor changes in the concentration ofintracellular calcium. In brief, medium containing an unknownconcentration of VEGF is added to an aliquot of suspended HUVEC,pre-loaded with Fura-2. Changes in fluorescence representing changes inintracellular calcium release are measured using a Hitachi 2000° F.fluorometer. Positive (histamine, thrombin) and negative (EGTA) controlsare also analyzed. This method is extremely sensitive and has a limit ofdetection of 0.2 pM.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 53                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGCCGGGCCGCCAGCACACT20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGCCGGGCCGCCAGCACACU20                          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acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAAGACAGCAGAAAGTTCAT20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CACCCAAGACAGCAGAAAG19                                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CACCCAAGACAGCAG15                                                             (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCAATGCACCCAAGACAGCAGAAAG25                                                   (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ACGCACACAGAACAAGACG19                      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                                                (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GTTCATGGTTTCGGAGGCCC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GTGCAGCCTGGGACCACTTG20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGCCTCGGCTTGTCACATCT20                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CTTCCTCCTGCCCGGCTCAC20                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CUUCCUCCUGCCCGGCUCAC20                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CTTCCTCCTGCCCGG15                                                             (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGCTCCTTCCTCCTGCCCGGCTCAC25                                                   (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GTCTCCTCTTCCTTCATTTC20                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GTCTCCTCTTCCTTC15                                                             (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GCAGAGTCTCCTCTTCCTTCATTTC25                                                   (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CGGACCCAAAGTGCTCTGCG20                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CCAAAGTGCTCTGCG15                                                             (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCCTCCGGACCCAAAGTGCTCTGCG25                                                   (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GGGCACGACCGCTTACCTTG20                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGGACCACTGAGGACAGAAA20                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CACCACTGCATGAGAGGCGA20                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TCCCAAAGATGCCCACCTGC20                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CGCATAATCTGGAAAGGAAG20                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ACTTTCTGCTGTCTTGGGTG20                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CATGGTTTCGGAGGCCCGAC20                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA/RNA                                                  (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CAUGGTTUCGGAGGCCCGAC20                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TTCATGGTTTCGGAGGCCCG20                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GACCGCTTACCTTGGCATGG20                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CCTGGGACCACTGAGGACAG20                                                        (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GGGACTCACCTTCGTGATGA20                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GAACTTCACCACTGCATGAG20                                                        (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TCCCAAAGATGCCCACCTGC20                                                        (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GCATAATCTGGAAAGGAAGG20                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      ACATCCTCACCTGCATTCAC20                                                        (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      ACATCCUCACCTGCAUUCAC20                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TTTCTTTGGTCTGCAATGGG20                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GGCCACTTACTTTTCTTGTC20                                                        (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CACAGGGACTGGAAAATAAA20                                                        (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GGGAACCAACCTGCAAGTAC20                                                        (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GTCACATCTGAGGGAAATGG20                                                        (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      CAGCCTGGCTCACCGCCTTGG21                                                       (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TCGCGCTCCCTCTCTCCGGC20                                                        (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CATGGTTTCGGAGGGCGTC19                                                         (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TCCGAAACCATGAACTTTCTG21                                                       (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GGTTTCGGAGGCCCGACCG19                                                         (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      CAAGAGAGCAGAAAGTTCAT20                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      CACCCAAGAGAGCAGAAACT20                                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      TCGTGGGTGCAGCCTGGGAC20                                                        __________________________________________________________________________

What is claimed is:
 1. A method of inhibiting VEGF gene expression in amammal comprising the step of administering intravitreally to an eye ofa mammal an effective VEGF gene expression-inhibiting amount of asynthetic oligonucleotide which is complementary to VEGF mRNA, theoligonucleotide having a nucleotide sequence selected from the groupconsisting SEQ ID NOs: 1, 2, 6, 14, 15, 18, 21, 28, 30, 31, and 32.